Since interferon' per se shows little promise as a prophylactic or therapeutic agent, interest has shifted to a search for acceptable inducers by which the body might be stimulated to make its own interferon.Shope2 demonstrated that a substance which was derived from Penicilliumfuniculosum and which was called helenine induced resistance to Semliki Forest and to Columbia SK virus infections in mice. Lewis et al.3 fractionated helenine and prepared a factor active in the mouse assay which exhibited properties of a nucleoprotein. Rytel, Shope, and Kilbourne4 demonstrated that helenine elicited a viral inhibitor in cell cultures and in mice which exhibited properties similar to those of interferon. In commenting, they stated that "the active principle in helenine is still a matter of conjecture since it has not been obtained in a sufficiently pure form to permit definite identification." Studies in our laboratories during the past several years have been directed toward the discovery of an interferon inducer which would be worthy of clinical evaluation. It was found that certain ribonucleic acids were highly active in inducing interferon and host resistance but were dependent upon (a) freedom from inhibitory protein and (b) multistrandedness of the RNA. Ribonucleoproteins and single-stranded nucleic acids were inactive.This series of papers describes the purification or synthesis and characterization of three kinds of multistranded RNA active in inducing interferon and host resistance. The present report describes the isolation and characterization of a double-stranded RNA from extracts of Penicillium funiculosum (helenine) which, when freed of protein, is a highly active inducer of interferon and host resistance. The substance is referred to as HeI-RNA. Subsequent papers will present data showing high level interferon and host resistance-inducing activities of multistranded complexes of synthetic polynucleotides5 and of double-stranded RNA of reovirus origin.6 Materials and Methods.-(1) Extract of Penicitlium funiculosum:7 P. funiculosum was grown as described elsewhere.3 The mycelium was extracted with 0.033 M sodium phosphate buffer, pH 8.0, by rapid stirring for 1 hr at room temperature. The clarified extract was treated as shown in steps A-C of Table 1. The supernate served as starting material for fractionation and purification purposes.(2) Assay for interferon induction in rabbits: Young adult New Zealand white rabbits weighing 4-5 lb were injected intravenously with 0.5 ml of the material for assay. Blood samples were taken 2 hr later. The sera were assayed for interferon content in primary rabbit renal cell cultures in roller tubes or in plaque assay flasks by published procedures8 9 using vesicular. stomatitis virus for challenge. The titer of interferon was the highest initial dilution of rabbit serum which showed 100% suppression of viral cytopathic effect in 50(% or more of the tube cultures, or which effected at least 50% reduction in plaques compared with controls in the plaque assay. The plaque assay...

468PURIFICATION AND CHARACTERIZAT'ION OF INTERFERON sorption of HC03-ions per se, but rather is the secretion of H+ ions which react with filtered HC03-to generate H2C03. Second, the accumulation of excess H2C03 in tubular fluid disproves the previously suggested hypothesis (3,7) that endogenous carbonic anhydrase might be located in the luminal membrane of renal tubular cells, thereby exerting a catalytic action on dehydration of excess H2C03 in tubular fluid.Finally, the calculated pH gradient (pHB-pHTE') between blood and tubular fluid was 1.07. For a pH gradient of this magnitude to result from passive distribution of H+ ions along an electrochemical gradient (8,9), it can be calculated from the Nernst equation [ET = -61.5 (pHB-pHTF)] that a transtubular potential difference ( ET) greater than 66 mV, lumen negative to blood, would be required. Previous measurement of proximal ET under conditions of NaHC03 diuresis gave an average value of only -20 mV( 10). Therefore, it is concluded that H+ ions are secreted into the lumen against an electrochemical gradient by an active transport process.Various virus-host animal or virus-cell culture systems have been shown to elaborate a "soluble" substance called interferon ( 1 ) , which interferes with the propagation of viruses in related host or host cell systems. Interferon appears to be an active principle in the virus interference phenomenon. Interferon, as first described by Isaacs and Lindenmann( l ) ? was prepared in fragments of chick chorioallantoic membrane incubated with hea t-inac tivated influenza virus. Wagner ( 2 ) demonstrated the presence of an interferon in the allantoic fluid of embryonated eggs infected with influenza virus and other workers have demonstrated interferons in other systems. Several groups of workers (2-6) have described the biological and biophysical properties of crude or semi-purified interferons. The present report describes the biological and biophysical qualities of a highly purified interferon derived from the allantoic fluid of embryonated hens' eggs infected with the WS strain of influenza virus.Materials and methods. Preparation of crude interferon. Nine-or 10-day-old embryonated eggs were inoculated by the allantoic route with lW5 ID50 of WS influenza strain RIE 5372 virus obtained from Dr. I. Tamm. The eggs were incubated at 36°C for 84-96 hours after which they were chilled a t 4OC and the allantoic fluid containing the interferon was harvested. The crude fluids were centrifuged in the cold for 20 minutes at 1500 rpm and the supernates were stored at 4°C for periods up to 14 days prior to processing for purification. In vitro assay for interferon activity. Serial 2-fold dilutions of the interferon sample were diluted in mixture 199 containing 1% calf serum and antibiotics, and one ml amounts were added in by guest on August 11, 2015 ebm.sagepub.com Downloaded from

Major effort has been given in our laboratories to finding a highly active inducer of interferon which would be worthy of clinical evaluation in man and domestic animals. Recent reports'-3 by our group recorded induction of interferon and resistance to viral infection in vitro and in vivo by complexed synthetic polynucleotides (I:C), by RNA derived from an extract of Penicillium funiculosum (Hel-RNA), and by RNA from the virion of reovirus type 3 (Reo 3-RNA). The essential property of RNA required for such induction was found to be doubleor multistranding and, in certain instances, freedom from inhibitory substances. The present report describes a double-stranded RNA extracted from E. coli infected with MS2 coliphage which was highly active as an inducer of interferon and resistance to viral infections. The substance, called MS2-RF-RNA, is of special promise as a practical source of double-stranded RNA for clinical purpose. Materials and Methods.-MS2 coliphage and E. coli strain Hfr 3000 were obtained from Dr. A. J. Clark of the Molecular Biology and Virology Laboratory, University of California, Berkeley. The phage was propagated and assayed quantitatively according to methods described by Loeb and Zinder.4 Preparation of double-stranded replicative form of MS2 coliphage RNA (MS2-RF-RNA): The methods described by Weissmann et al.5 and Billeter et al.6 wereused. E. coli cultures in exponential growth phase were infected with MS2 at an input multiplicity of about ten and incubated for one hour at 37°C with continuous aeration. The collected cells from one liter of culture medium were lysed using sodium dodecyl sulfate and the lysate was repeatedly extracted with phenol to remove protein. The aqueous phase was then treated with RNase and DNase, and the RNA was separated from smaller molecular weight substances by exclusion chromatography employing Sephadex G-200. The further purification including final removal of the bacterial substances is described in the text.Preparation of single-stranded MS2 virion form (VF) RNA (MS2-VF-RNA): The E. coli cultures were infected with MS2 phage and incubated overnight at 37°C with aeration. Cell lysis was aided by further incubation at 37°C for 30 minutes with lysozyme and ethylenediaminetetraacetate (EDTA) in final concentrations of 50 ,ug/ml and 0.1 M, respectively. The clarified lysate contained 3.8 X 1011 plaque-forming units of phage per milliliter. The virus was concentrated by the acid-precipitation method of Charney et al., I resuspended in phosphate-buffered saline, and purified further by freon extraction and density-gradient centrifugation in CsCl according to Strauss and Sinsheimer.8 The pooled virus from the gradient (1.8 X 1013 PFU/ml) was repeatedly dialyzed against 0.02 M phosphate buffer at pH 7.0. The final virus preparation showed a typical ultra-2102

Use of metabolic inhibitors provides an effective approach to the study of metabolic requirements of virus reproduction (1). In the area of nucleic acid metabolism it has been shown that inhibition of host deoxyribonucleic acid (DNA) synthesis does not inhibit the multiplication of Newcastle disease (2-4) or poliomyelitis virus (2). Thus, host DNA synthesis is apparently not required for the production of several ribonucleic acid (RNA)-containing viruses; whether preformed DNA is necessary is not known.The present study is concerned with the question whether host RNA plays a role in the reproduction of DNA-containing viruses. This question was explored with the aid of 5,6-dichloro-1-/3-~ribofuranosylbenzimidazole (DRB) 1 and with ribonuclease (RNase).In this communication the inhibitory effects of DRB on adenosine-8-C n uptake into cell RNA, on C14-L-alanine uptake into cell proteins, and on cellular oxygen consumption are described. Blocking of the inhibitory effect of DRB on influenza virus multiplication by adenosine will be reported. In addition, the inhibitory activity of DRB on adenovirns multiplication will be compared with its effect on influenza, poliomyelitis, and vaccinia virus multiplication (5-7).In a second communication (8) results of studies of the effects of RNase on influenza B and vaccinla virus multiplication in the chorioallantoic membrane, and on pock formation by vaccinla and herpes simplex viruses will be described

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